Cell death programmed that the cell itself is to positively bring about death under the physiological condition, namely apoptosis, is known to be a mechanism equipped to a living body in order to remove aging cells in immune system and unfavorable cells for the living body such as morbid cells. Such apoptosis is characterized in rapid contraction in cell size and change in a cell nucleus, apoptotic cells usually become apoptotic bodies and are to be finally engulfed by phagocytes such as macrophages and the like. For instance, it is well known that cells first contract and detach from adjacent cells, a chromatin which is a complex of DNA of nucleus and protein is compressed around a nuclear membrane to cause concentration of nucleus, and microvillus on the cell surface is vanished and smoothed at the same time, a protuberances of various sizes appear and they will gradually be constricted and torn apart, then fractionated into globular apoptotic bodies of various sizes enveloped in membrane, and such bodies are engulfed and eliminated by macrophages or adjacent phagocytes.
In the meantime, synthetic materials such as aminopterin, methotrexate, 8-azaguanine, 6-mercaptopurine, 5-fluorouracil, 1-(2-tetrahydrofuryl)-5-fluorouracil, etc., and antibiotics such as mitomycin C, chromomycin, bleomycin, etc., interferon, CSF inhibitor, CBF, etc., are known to be used to inhibit the proliferation of morbid cells such as cancer cells and malignant tumor cells and to treat diseases resulting from these cells. All of them act to a certain cell and cause necrosis to remove morbid cells. Unlike necrosis, which occurs by pathological factor, apoptosis is known to occur not only by pathological factor but also by various physiological factors.
It is reported that apoptosis is accompanied by sequence change of the cell membrane phospholipids which comprise a cell in the early stage, and results in the exposure to the cell surface of phosphatidylserine which is a negatively-charged phospholipid (Immunol. Today, 14:131-136, 1993; Cirk. Res., 77:1136-1142, 1995). It is considered that these changes on the cell surface are recognized by macrophages and adjacent cells, and phagocytic stages proceed. It is considered that the exposure of phosphatidylserine to the cell surface of apoptotic cells plays an important role in phagocytic mechanism since the above-mentioned phagocytic stages are inhibited by annexin V which selectively binds to phosphatidylserine (Biochem. Biophys. Res. Commun., 205:1488-1493, 1994; Proc. Natl. Acad. Sci. USA, 93:1624-1629, 1996). Besides, detection of early stage of apoptosis is conducted by flowcytometry using labeled body of annexin V.
On the other hand, MFG-E8: milk fat globule-EGF factor 8 is cloned as a secretory protein derived from mammary epithelia abundantly contained in breast milk (Biochem. Biophys. Res. Commun. 254 (3), 522-528, 1999), which is known as a secretory glycoprotein strongly expressing in many other normal tissues or several tumor cells afterwards. MFG-E8 is comprised of two EGF (epidermal growth factor) domains from N termini side and a domain which has homology with C1 and C2 domains of a blood coagulation factor V and VIII. Homology of MFG-E8 is reported in several mammals including humans (BA46, lactadherin), mice (MFG-E8), rats (rAGS), pigs (P47), cows (PAS-6, PAS-7), and endothelial cell-specific cell adhesion molecule DELI which has similarity in domain structure with MFG-E8 has been cloned, further, MFG-E8 and DELI contain RGD sequence which binds to integrin in their second EGF domain. Besides, C1 and C2 domains of C termini side are known to bind to phospholipids on cell membrane. However, many points regarding the relation with enzymatic activity and its physiological function of MFG-E8 are still unknown. In order to clear these points, genomic gene of mouse milk fat globule-EGF factor 8 MFG-E8 and chromosome mapping, kinetics of gene expression in development stage, intracellular localization and the like have been considered, and it is recognized that reproductive rudiment is a main expression part at the early development stage of MFG-E8, and there is a strong expression characteristic to neuron or cartilage rudiment at a later development stage. Further, attempts have been made to generate MFG-E8 gene deficient mouse in order to investigate the function of MFG-E8 in vivo.
Apoptosis plays an important role in maintaining the homeostasis of living body. It is necessary to remove apoptotic cells immediately by macrophages in order to protect normal cells from noxious substance secreted by the cells undergoing apoptosis (apoptotic cells). For instance, cancer can be treated by positively inducing apoptosis in cancer cells. Even in such case, however, it is necessary to remove apoptotic cells immediately. The object of the present invention is to provide a removal promoter for apoptotic cells which can immediately remove apoptotic cells in vivo by macrophages, and a removal inhibitor which inhibits the removal of apoptotic cells in vivo by macrophages.
As a result of keen study in order to solve the above-mentioned issues, the present inventors found that milk fat globule-EGF factor 8 (MFG-E8-L) binds specifically to apoptotic cells by recognizing aminophospholipid such as phosphatidylserine (PS) and the like which are exposed on the cell surface once the cells started to move toward apoptosis, and MFG-E8-L promote the phagocytosis of apoptotic cells by macrophages, and that D89E mutant which is a point mutant derivative of MFG-E8-L inhibits the phagocytosis of apoptotic cells by macrophages. Thus, the present invention has been completed.